Effects of spermine on the detection of induced forward mutation at the Can1 locus in yeast: evidence for selection against canavanine-resistant mutants.

The effect of exogenous spermine tetrahydrochloride (0.5 mg/ml) on hydrazine- and nitrous acid-induced forward mutation to canavanine resistance (CAN1 leads to can1, normal to defective arginine permease) was examined in stationary-phase haploid Saccharomyces cerevisiae. Post-treatment cell division (specifically DNA replication) is required for hydrazine mutagenesis at this locus, whereas nitrous acid mutagenesis exhibits, in addition, a significant post-treatment-independent component. Spermine addition only during mutagenic treatments in buffer did not affect mutagen cytotoxicity, but did result in a slight yet consistent decrease in induced mutation frequencies. Addition of spermine to the yeast extract--peptone--dextrose (YEPD) post-treatment growth medium resulted in dramatic reductions of induced mutation frequencies, which could be alleviated by pregrowth in spermine-containing YEPD. Such a medium was found to cause an apparent temporary growth inhibition for almost 40 h, after which the growth rate of the culture increased rapidly. Cultures "recovering" from spermine inhibition were no longer inhibitable by spermine in fresh medium, suggesting an outgrowth of spontaneous and/or induced spermine-resistant derivatives. Genetic analysis of one isolate revealed a single dominant nuclear gene conferring resistance by some means other than defective spermine uptake. Growth of this mutant was only slightly inhibited by spermine (20% increase in doubling time), while mutation expression remained high. Results of competitive growth experiments indicated that spermine-containing YEPD exerted a selection pressure against canavanine-resistant cells, while YEPD by itself did not. The mechanism for this selection is not presently understood. With respect to replication-dependent induced mutation at CAN1, our initial observation of a strong apparent antimutagenic action of spermine was found to be best explained by this specific selection against can1 mutants. This underscores the need for caution in the interpretation of experiments designed to study physiological modification of mutagenic potential.     

Mutagenesis of yeast by hydrazine: dependence upon post-treatment cell division.

Hydrazine was found to be mutagenic for yeast (Saccharomyces cerevisiae) at exposures (concentration × time) ranging over nearly three orders of magnitude. Little or no forward mutation from CAN1 to can1 was detectable upon immediate plating following treatment in neutral buffer suspension. Post-treatment cell division in yeast extract peptone dextrose complex growth medium was required for expression of induced mutation to canavanine resistance. Frequencies of induced mutation rose to levels approximately 10-fold higher than spontaneous levels for exposures between 0.1 and 12.0 min mol/l. Survival remained at 100%. For exposures greater than 80 min mol/l viability and mutation frequency began to decrease sharply. By contrast, single treatments of ethyl methanesulfonate, methyl methanesulfonate, N-methyl-N′-nitro-N-nitro-soguanidine, nitrous acid, hydroxylamine, and ultraviolet light were able to increase mutation frequency with this system upon immediate assay. Further growth-dependent increases in mutation frequency were not observed with HA and UV.Expression of HZ-induced mutation was detectable after treated cells had undergone less than one population doubling in YEPD. Such mutation expression could be blocked by the inhibitors cycloheximide and hydroxyurea, which block protein synthesis and DNA synthesis respectively. Results were similar to those obtained previously with Haemophilus influenzae and similarly suggest that, in this eukaryote, HZ-induced lesions lead to mutation by causing base mispairing at DNA replication rather than by means of an error-prone repair mechanism.